Project 3, Summary/Abstract The roles of noncoding (nc)RNAs in lymphoid cells harboring one of three transforming herpesviruses are being investigated. Epstein-Barr virus (EBV) infects and transforms human B cells; it is the causative agent of infectious mononucleosis and is associated with several human cancers. Herpesvirus saimiri (HVS) induces fatal lymphomas and leukemias in New World monkeys and transforms monkey T lymphocytes in culture. Kaposi's sarcoma-associated herpesvirus (KSHV) afflicts immunocompromised individuals and persists in a latent form until lytic activation. The two EBV-encoded EBERs, the newly discovered EBV stable intronic sequence (sis)RNA, seven HVS-encoded HSURs, and six HVS microRNAs are all expressed in virally transformed cells. Upon induction, KSHV produces PAN, a capped, polyadenylated nuclear RNA discovered in George Miller's laboratory. These viral ncRNAs are abundant, conserved and bind host proteins. Our functional studies have uncovered novel mechanisms of microRNA biogenesis and decay, identified the role of triple helices in RNA stabilization, and contributed important insights into viral evolution and host pathways perturbed during viral latency or lytic growth. Proposed aims will extend these advances to further elucidate molecular mechanisms. The contributions of HSURs1 and 2 and HVS microRNAs to T-cell activation, proliferation and immune surveillance are being validated. HVS microRNA processing will be scrutinized for the novel use of the host Integrator complex. Since PAN RNA is essential for KSHV late gene expression, we will ask whether its interaction with host poly(A) binding protein C1 (PABPC1) relocalized to the nucleus during lytic infection is essential and whether late viral mRNAs escape host shutoff by exiting the nucleus via a novel pathway involving nuclear envelope budding. Global approaches to EBER function have implicated them in post-transcriptional events in gene expression. EBER2 RNPs have been localized, along with the important B-cell transcription factor Pax5, at the terminal repeats of EBV episomes, suggesting a role in the maintenance of latency. The EBV sisRNA-1 is predicted to bind host microRNAs, leading to their sequestration or degradation. Structural studies of the KSHV PAN RNA stability element complexed with PABPC1 protein will provide molecular insights.